Stimulation of an immune response is dependent upon the presence of antigens recognized as foreign by the host immune system. The discovery of the existence of tumor associated antigens has now raised the possibility of using a host's immune system to intervene in tumor growth. Various mechanisms of harnessing both the humoral and cellular arms of the immune system are currently being explored for cancer immunotherapy.
Elements of the cellular immune response are capable of specifically recognizing and destroying tumor cells. The isolation of cytotoxic T cells (CTL) from tumor-infiltrating cell populations or from peripheral blood suggests that such cells play an important role in natural immune defenses against cancer (Cheever et al., Annals N.Y. Acad. Sci. 1993 690:101–112). CD8+ T cells (TCD8+) in particular, which recognize Class I molecules of the major histocompatibility complex (MHC)-bearing peptides of 8 to 10 residues derived from proteins located in the cytosols, are believed to play an important role in this response. There are now numerous examples of both mouse and human TCD8+ that specifically recognize tumor cells and have therapeutic activity after adoptive transfer, in some cases inducing complete remission. However, despite the potential for T cells to eradicate tumors, it is obvious from the progressive growth of most cancers that many tumors escape recognition by TCD8+ in vivo. The induction of sufficient T cells in vivo has been difficult. Though a variety of tumors have been found to be immunogenic, stimulation of an effective antitumor immune response has been difficult to demonstrate.
One explanation for this phenomena is that tumors may be capable of delivering antigen-specific signals to T cells, but not the co-stimulatory signals necessary for full activation of T cells. Co-stimulation of T cells occurs when a surface molecule, B7, on the presenting cells interacts with a T cell molecule known as CD28. It has been observed that T cells receiving the antigen-specific signal (but not B7) become unresponsive. Many tumor cells do not carry the B7 protein, therefore B7 has been added to cancer cells (Travis, J., Science 1993 259, 310–311). It has been demonstrated that expression of the co-stimulatory ligand B7 on melanoma cells induced the rejection of a murine melanoma in vivo (Townsend, S. E. and Allison, J. P., Science 1993, 259, 368–370). This rejection was found to be mediated by CD8+ T cells; CD4+ T cells were not required. These results suggest that B7 expression may render tumor cells capable of effective antigen presentation, resulting in their eradication in vivo.
The effects of localized secretion of cytokines on tumor progression has also been studied. Secretion of low levels of interleukin-2 (IL-2) in a mouse fibrosarcoma cell line transfected with the human IL-2 gene introduced via a retroviral vector was found to abrogate the tumorigenicity of these cells and induce a long lasting protective immune response against a subsequent challenge with a tumorigenic dose of parent cells (Gansbacher et al., J. Exp. Med. 1990, 172, 1217–1224). In another study, cells from a spontaneously arising murine renal cell tumor were engineered to secrete large doses of interleukin-4 (IL-4) locally (Golumbek et al., Science 1991, 254, 713–716). Animals injected with the tumor cells rejected the IL-4-transfected tumors in a predominantly T cell-independent manner. However, these animals developed a T cell-dependent systemic immunity to the parental tumor. The systemic immunity was tumor-specific and mediated by CD8+ T cells. These experiments suggest that it may be possible to cure parental tumors by generating a systemic immune response by the injection of genetically engineered tumor cells.
There is also evidence to suggest that some tumor cells express low levels of class I molecules in vivo and in vitro. Intracellular antigens must be processed before presentation to CD8+ T cells by major histocompatibility complex (MHC) class I molecules. The antigen processing efficiency of 26 different human tumor lines has been studied (Restifo et al., J. of Exp. Med. 1993, 177, 265–272). Three different cell lines, all human small cell lung carcinoma, consistently failed to process endogenously synthesized proteins for presentation to the T cells. Pulse chase experiments showed that MHC class I molecules were not transported by these cells lines from the endoplasmic reticulum to the cell surface. Northern blot analysis showed that these cells contained little or no mRNA encoding MHC-encoded proteosomes and transporter genes. Treatment with interferon γ enhanced expression of these mRNAs and reversed the observed functional and biochemical deficits. Thus, potential therapeutic applications which include enhancing antigen processing at the level of transcription of MHC-encoded proteosome and transporter genes was suggested.
Immunizing patients with recombinant BCG (bacille Calmette-Guèrin) or Salmonella bacteria carrying a gene coding for an antigenic peptide has also been suggested as an oral tumor immunotherapy (Boon et al. Annu. Rev. Immunol. 1994, 12, 337–65). Orally administered live attenuated Salmonella recombinant vaccine, which expressed the full length P. berghei circumsporozite antigen, has been shown to protect mice against malaria. This immune response was mediated by the induction of CD8+ T cells (Aggarwal et al., J. of Exp. Med. 1990, 172, 1083–1090). It is suggested that live attenuated Salmonella recombinants may be useful in the study of other diseases where CTL-mediated immunity may be important. However, no other experiments were reported. BCG has also been implicated as a novel live-vaccine vehicle which may prove useful in stimulating both humoral and cellular immune response to a wide variety of viral, bacterial and protozoal antigens (Stover et al., Nature 1991, 351, 456–460).
It has now been found that the immune response to an antigen, and in particular a tumor associated antigen, can be induced by the administration of a vaccine comprising a listeriolysin fusion protein comprising a tumor associated antigen or a recombinant form of the intracellular bacterium Listeria monocytogenes which expresses a tumor associated antigen or fragment thereof. The recombinant form of Listeria monocytogenes can express the tumor associated antigen alone or as a listeriolysin fusion protein which comprises the tumor associated antigen. In one embodiment, one or more vectors comprising recombinant Listeria monocytogenes each expressing a different tumor associated antigen or fusion protein thereof, can be used in a vaccine to stimulate an immune response. In this embodiment, it is preferred that the expressed tumor associated antigens be fused to listeriolysin. In another embodiment, one or more fusion proteins, each fusion protein comprising a truncated form of listeriolysin fused to a different tumor associated antigen, can be used. As demonstrated herein, administration of the vaccines of the present invention decrease the size of existing tumors and inhibit formation of primary tumors. No other stimulation following antigen presentation was required to induce this response.